The long term goal of my research program is to understand mechanisms of constitutive and regulated pre-mRNA 3'end processing. Two types of sequence elements constitute viral and cellular polyadenylation signals. Core elements, consisting of conserved upstream AAUAAA and downstream U-rich motifs, mediate basal levels of 3'end processing. Relatively uncharacterized auxiliary elements, located upstream or downstream of the core elements, may act as enhancers or repressors of 3'processing. Many regulated polyadenylation signals which are used selectively contain either (1) non-consensus sequences in place of the core elements or (2) novel auxiliary elements located upstream or downstream of the core elements. The focus of this proposal will be to elucidate mechanisms of selective polyadenylation of viral pre-mRNAs by identifying functional alternative core motifs and auxiliary elements and characterizing factors which interact with them. First, we will identify and characterize alternative sequence motifs which can serve as core downstream elements by mapping the site of CstF interaction on the unconventional Human Papillomavirus (HPV) 11, 16 and 35 early polyadenylation signals. Second, using a systematic series of mutations, we will identify the core upstream elements of the regulated HPV 11 early polyadenylation signal which lacks a conventional AAUAAA. Third, using a similar series of directed mutations, we will identify novel auxiliary upstream and downstream elements in these three signals. Proteins which specifically bind these elements will be characterized by a UV cross linking approach. Fourth, we will investigate the mechanism of stimulation of 3'end processing by DSEF-1, the only known example of an auxiliary downstream element binding factor. Collectively, these first four studies will systematically characterize nucleic acid elements and protein factors which may play role in selective usage of polyadenylation signals and provide mechanistic insights into the role of these elements in the regulation of HPV 3'end formation. Finally, we will apply the insights gained in these studies to elucidate the mechanism of selective usage of B19 parvovirus poly(A) signals at early and late times post infection. The proposed studies will, therefore, lead to an understanding of a key process in viral gene expression. This information, in turn, will provide an understanding at the molecular level of factors which influence viral host range and pathogenesis, as well as identify potential areas to target viral therapeutics.